Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle

Abstract

The muscle LIM protein (MLP) and cofilin 2 (CFL2) are important regulators of striated myocyte function. Mutations in the corresponding genes have been directly associated with severe human cardiac and skeletal myopathies, and aberrant expression patterns have often been observed in affected muscles. Herein, we have investigated whether MLP and CFL2 are involved in common molecular mechanisms, which would promote our understanding of disease pathogenesis. We have shown for the first time, using a range of biochemical and immunohistochemical methods, that MLP binds directly to CFL2 in human cardiac and skeletal muscles. The interaction involves the inter-LIM domain, amino acids 94 to 105, of MLP and the amino-terminal domain, amino acids 1 to 105, of CFL2, which includes part of the actin depolymerization domain. The MLP/CFL2 complex is stronger in moderately acidic (pH 6.8) environments and upon CFL2 phosphorylation, while it is independent of Ca 2؉ levels. This interaction has direct implications in actin cytoskeleton dynamics in regulating CFL2-dependent F-actin depolymerization, with maximal depolymerization enhancement at an MLP/ CFL2 molecular ratio of 2:1. Deregulation of this interaction by intracellular pH variations, CFL2 phosphorylation, MLP or CFL2 gene mutations, or expression changes, as observed in a range of cardiac and skeletal myopathies, could impair F-actin depolymerization, leading to sarcomere dysfunction and disease.The muscle LIM protein (MLP) has emerged as a critical player in striated muscle physiology and pathophysiology over recent years. MLP, encoded by the CSRP3 (or CRP3) gene, is a member of the conserved LIM-only protein family since it contains two LIM functional zinc finger domains, and it is expressed exclusively in muscle cells. Specifically, in differentiating striated muscle cells, MLP localizes in the nucleus, promoting myogenic differentiation (8), while in adult muscle, it translocates to the cytoplasm and assumes an essential role in myocyte cytoarchitecture. Through its interactions with structural proteins, such as alpha-actinin, telethonin (T-cap), ␤I-spectrin, and N-RAP, MLP has been suggested to act as a scaffold protein for the basic contractile unit, the sarcomere, and the actin-based cytoskeleton (7,9,25,27,46,73).Mutations in CSRP3 have been directly associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathies (9,31,32,46). HCM is the most common genetic myocardial disease, with a prevalence of 0.2% in adults, and the most frequent cause of sudden cardiac death in young individuals, while DCM is the third most common cause of heart failure (54,55,76). Genetic aberrations in MLP have been shown to lead to marked actin cytoskeleton disorganization and disrupted cardiac myofibrillar cytoarchitecture (9). Similar observations have been described for skeletal muscles, and HCM-CSRP3 mutations have been associated with mild skeletal myopathy (9, 31). Various hypotheses, including an MLP role in mechanical stretch sensing or the mechanical stress response, have...

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

“…S4B and C). In contrast to the findings of Papalouka et al (44), who reported that at specific MLP/cofilin ratios MLP enhances CFL2 depolymerizing activity, our data support that CFL2 and MLP have opposite effects on AF dynamics irrespective of their relative concentrations.…”

“…In addition, MLP-GFP colocalized with ␣-ac- tinin in nascent myofibrils (Fig. 3D to G), an observation that is well consistent with the previously reported localization of endogenous MLP at the Z line of muscle cells (18,24,44,47). In support of MLP also functioning as an actin cross-linker/stabilizer in differentiated muscle cells/tissues, MLP-GFP-expressing HL-1 cells frequently exhibited more prominent actin-based structures than the surrounding nontransfected cells (Fig.…”

“…Full AF stabilization occurred at MLP/actin molar ratios of Ͼ1:1. Since MLP was recently shown to colocalize with cofilin 2 (CFL2) at the sarcomeric Z line of human striated muscles and to directly interact with CFL2 in vitro (44), additional pyrene-actin depolymerization assays were conducted in the presence of CFL1 or CFL2. Results indicate that MLP strongly reduces the ability of CFL1 and CFL2 to enhance actin depolymerization, supporting that MLP protects AF from depolymerization (see Fig.…”

Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy

“…CFL2 is a muscle-specific protein that belongs to the actin depolymerization factor/cofilin family; it regulates actin dynamics by promoting actin filament turnover (51,53). CFL2 binds actin; phosphorylation prevents actin binding (7).…”

Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress

“…60 DFD meat occurs when the ultimate pH post-mortem measured after 12- 48 61 hours is higher than 6.0 [8]. The elevated pH is associated with relatively little 62 denaturation of proteins, water is tightly bound and little or no exudates are expelled 63 [6].…”